In view of the known biochemical mechanism of folic acid coenzyme systems, the homeosteric replacements of one of the nitrogen atoms in the pyrazine moiety by oxygen should produce effective antimetabolites; i.e., derivatives possessing the pyrimido (4,5-b) (1,4)oxazine and the pyrimido(5,4-b)(1,4) oxazine ring systems. The synthesis of such analogs of the vitamin will permit a study of the molecular binding in the various enzymic systems associated with the coenzyme structures. Further, the pyrimido(5,4-b)(1,4)oxazine nucleus does not have an N-5 position for single carbon transfer reactions, and may thus be of potential interest as a chemotherapeutic agent. At the molecular level, it is the pyrazine moiety which is directly involved in covalent reactions with single carbon units which ultimately permits the various enzymic reactions of the molecule. Further, prior to performing its catalytic function, this portion of the molecule must be reduced to a tetrahydro form. Homeosteric substitution studies in this area have been confined primarily to a carbon for nitrogen exchange, but the homeosteric replacement of nitrogen by oxygen in the pyrazine moiety of folic acid should produce a more isoelectronic structural analog. For comparative biological evaluations, the effect of these derivatives will be examined in microbiological assays and tissue culture systems as well as for their ability to inhibit or replace the coenzyme activity in folate reductase and thymidylate synthetase.